Structure-function analysis of the nsp14 N7-guanine methyltransferase reveals an essential role in Betacoronavirus replication (preprint)

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their mRNAs, protect them from degradation by cellular 5- exoribonucleases, and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bi-functional replicase subunit harboring an N-terminal 3-to-5; exoribonuclease (ExoN) domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome-CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

Impact of human airway epithelial cellular composition on SARS-CoV-2 infection biology (preprint)

Infection biology and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), are incompletely understood. Here, we assessed the impact of airway epithelial cellular composition on infection in air-liquid interface (ALI) cultures of differentiated primary human tracheal (PTEC) and bronchial epithelial cells (PBEC). We first compared SARS-CoV-2 infection kinetics, related antiviral and inflammatory responses, and viral entry factors in PTEC and PBEC. Next, the contribution of differentiation time was investigated by differentiating ALI-PTEC/PBEC for 3-5 weeks and comparing dynamics of viral replication/spread, cellular composition and epithelial responses. We observed a gradual increase in viral load with prolonged culture duration. Ciliated and goblet cells were predominantly infected in both PTEC and PBEC. Immunofluorescence analysis and RT-qPCR showed that compared to other cell types mainly ciliated and goblet cell numbers were affected by increased culture duration. An increased proportion of these two target cell types was associated with increased viral load. Furthermore, modulation of cellular composition using IL-13 and the Notch signaling inhibitor DAPT, underlined the importance of both ciliated and goblet cells for infection. DAPT treatment resulted in a lower viral load and a relative increase in ciliated cells at the expense of goblet cells, compared to IL-13 treated cultures in which both cell types were present and viral load was higher. In conclusion, our results identify cellular composition as a contributing factor to airway epithelial susceptibility to SARS-CoV-2. IMPORTANCEIn this study, we determined an effect of culture duration and airway cellular composition of ALI-PBEC and ALI-PTEC cultures on SARS-CoV-2 infection. We found that SARS-CoV-2 infection was increased with prolonged cell culture time and the total percentage and proportion of ciliated and goblet cells played an important role in infection level, suggesting that airway epithelial differentiation/maturation levels may in part determine susceptibility of SARS-CoV-2 infection. The development of effective therapies either targeting virus replication or pathogenesis against SARS-CoV-2 requires robust cell culture-based infection models to test small molecules and biologicals. Therefore, it is important to identify factors that are essential for reliably modeling SARS-CoV-2-airway epithelial cell interactions. This study sheds light on virus-airway epithelial cell interactions and adds to the complexity of SARS-CoV-2 cell tropism in the airways. In addition, the effect of IL-13 on viral infection hints at a causal connection between SARS-CoV-2 infection and (allergic) asthma.

6',6'-Difluoro-aristeromycin is a potent inhibitor of MERS-coronavirus replication (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the lack of treatments to combat infections with human or (potentially) zoonotic CoVs. Thus, it is critical to develop and evaluate antiviral compounds that either directly target CoV functions or modulate host functions involved in viral replication. Here, we demonstrate that low-micromolar concentrations of 6',6'-difluoro-aristeromycin (DFA), an adenosine nucleoside analogue, strongly inhibit the replication of Middle East respiratory syndrome coronavirus (MERS-CoV) in a cell-based infection assay. DFA was designed to target S-adenosylhomocysteine (SAH) hydrolase and, consequently, may affect intracellular levels of the methyl donor S-adenosylmethionine, which is used by two CoV methyltransferases involved in the capping of the 5 end of the viral mRNAs. Passaging of wild-type MERS-CoV in the presence of DFA selected a mutant with a ~100-fold decreased DFA sensitivity. This drug-resistant population carried various amino acid substitutions in the viral nonstructural proteins (nsp), including mutations in nsp16, which has 2-O-methyltransferase activity, and nsp13, which contains a nucleoside triphosphate hydrolase activity that has also been implicated in CoV capping. Based on our results, we hypothesize that DFA directly or indirectly affects viral cap methylation, either by inhibiting the viral enzymes involved or by binding to SAH hydrolase. We also evaluated the antiviral activity of DFA against other betacoronaviruses, but found it to have limited impact on their replication, while being quite cytotoxic to the Calu-3 cells used for this comparison. Nevertheless, our results justify the further characterization of DFA derivatives as an inhibitor of MERS-CoV replication.

Ad26.COV2.S-elicited immunity protects against G614 spike variant SARS-CoV-2 infection in Syrian hamsters and does not enhance respiratory disease in challenged animals with breakthrough infection after sub-optimal vaccine dosing (preprint)

Previously we have shown that a single dose of recombinant adenovirus serotype 26 (Ad26) vaccine expressing a prefusion stabilized SARS-CoV-2 spike antigen (Ad26.COV2.S) is immunogenic and provides protection in Syrian hamster and non-human primate SARS-CoV-2 infection models. Here, we investigated the immunogenicity, protective efficacy and potential for vaccine-associated enhanced respiratory disease (VAERD) mediated by Ad26.COV2.S in a moderate disease Syrian hamster challenge model, using the currently most prevalent G614 spike SARS-CoV-2 variant. Vaccine doses of 1x109 vp and 1x1010 vp elicited substantial neutralizing antibodies titers and completely protected over 80% of SARS-CoV-2 inoculated Syrian hamsters from lung infection and pneumonia but not upper respiratory tract infection. A second vaccine dose further increased neutralizing antibody titers which was associated with decreased infectious viral load in the upper respiratory tract after SARS-CoV-2 challenge. Suboptimal non-protective immune responses elicited by low-dose A26.COV2.S vaccination did not exacerbate respiratory disease in SARS-CoV-2-inoculated Syrian hamsters with breakthrough infection. In addition, dosing down the vaccine allowed to establish that binding and neutralizing antibody titers correlate with lower respiratory tract protection probability. Overall, these pre-clinical data confirm efficacy of a 1-dose vaccine regimen with Ad26.COV2.S in this G614 spike SARS-CoV-2 virus variant Syrian hamster model, show the added benefit of a second vaccine dose, and demonstrate that there are no signs of VAERD under conditions of suboptimal immunity.

Immunogenicity of one- and two-dose regimens of the Ad26.COV2.S COVID-19 vaccine candidate in adult and aged rhesus macaques (preprint)

The development of preventive corona virus disease (COVID)-19 vaccines is an urgent need, especially for the aging population that is most affected by the ongoing pandemic. The Janssen Ad26.COV2.S vaccine candidate is currently the only one evaluated as a single dose vaccination regimen in Phase 3 clinical studies. While the advantages of single dose vaccines, especially for use during a pandemic, are obvious, multiple doses may potentially improve magnitude and durability of immune responses. Here we assessed the immunogenicity of one- and two-dose Ad26.COV2.S vaccine regimens in adult and aged non-human primates (NHP). A second vaccine dose, administered 8 weeks post the first immunization, induced a significant increase in antigen-specific binding and neutralizing antibody responses in both adult and aged animals as compared to a single dose. In addition, in one-dose regimens neutralizing antibody responses were maintained for at least 14 weeks, providing an early indication of durable immune responses elicited by Ad26.COV2.S. Similar to what we showed previously in adult animals, Ad26.COV2.S vaccination of aged NHP induced a CD8+ T cell response and a Th1 skewed CD4+ T cell response. These data support the initiation of a two-dose Ad26.COV2.S regimen in a Phase 3 clinical trial in adults and elderly.

Impact of COVID-19: Decrease in the Number of Fledging Barn Swallow Chicks in Tokyo (preprint)

Barn swallows that have crossed the sea from Southeast Asia usually appear in the Kyushu Region of Japan around March after passing through Okinawa Prefecture. When the climate becomes warmer, these birds then move further north, nesting and raising their chicks in various parts of Japan. It is worth noting that barn swallows typically nest on man-made objects, for example, the roofs of houses and barns. It is believed that this is because barn swallows protect their eggs and chicks from foreign enemies such as sparrows and crows so they build their nests in populated areas. The barn swallows behavior of using the presence of people to keep foreign enemies away shows that barn swallows are quite wise. However, it has been reported that from the spring to summer of 2020, barn swallows, nesting and raising their chicks, which were seen every year, were not found in various parts of Japan. Therefore, we investigated the relationship between peoples self-restraint from going out and the fledging of barn swallow chicks in Tokyo metropolitan during the corona virus disease 2019 (COVID-19) era. The results of the survey showed a link between peoples refraining from going out and the fledging of barn swallow chicks. Next spring of 2021, the termination of COVID-19 is an important environment for swallow chick fledging.

Ad26-vector based COVID-19 vaccine encoding a prefusion stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses (preprint)

Development of effective preventative interventions against SARS-CoV-2, the etiologic agent of COVID-19 is urgently needed. The viral surface spike (S) protein of SARS-CoV-2 is a key target for prophylactic measures as it is critical for the viral replication cycle and the primary target of neutralizing antibodies. We evaluated design elements previously shown for other coronavirus S protein-based vaccines to be successful, e.g. prefusion-stabilizing substitutions and heterologous signal peptides, for selection of a S-based SARS-CoV-2 vaccine candidate. In vitro characterization demonstrated that the introduction of stabilizing substitutions (i.e., furin cleavage site mutations and two consecutive prolines in the hinge region of S1) increased the ratio of neutralizing versus non-neutralizing antibody binding, suggestive for a prefusion conformation of the S protein. Furthermore, the wild type signal peptide was best suited for the correct cleavage needed for a natively-folded protein. These observations translated into superior immunogenicity in mice where the Ad26 vector encoding for a membrane-bound stabilized S protein with a wild type signal peptide elicited potent neutralizing humoral immunity and cellular immunity that was polarized towards Th1 IFN-{gamma}. This optimized Ad26 vector-based vaccine for SARS-CoV-2, termed Ad26.COV2.S, is currently being evaluated in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT04436276).

The enzymatic activity of the nsp14 exoribonuclease is critical for replication of Middle East respiratory syndrome-coronavirus (preprint)

AO_SCPLOWBSTRACTC_SCPLOWCoronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains an N-terminal 3-to-5 exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase) domain. While the latter presumably operates during viral mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN-knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously found to have a crippled but viable hypermutation phenotype. Remarkably, using an identical reverse genetics approach, an extensive mutagenesis study revealed the corresponding ExoN-knockout mutants of another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), to be non-viable. This is in agreement with observations previously made for alpha- and gammacoronaviruses. Only a single MERS-CoV ExoN active site mutant could be recovered, likely because the introduced D191E substitution is highly conservative in nature. For 11 other MERS-CoV ExoN active site mutants, not a trace of RNA synthesis could be detected, unless - in some cases - reversion had first occurred. Subsequently, we expressed and purified recombinant MERS-CoV nsp14 and established in vitro assays for both its ExoN and N7-MTase activities. All ExoN knockout mutations that were lethal when tested via reverse genetics were found to severely decrease ExoN activity, while not affecting N7-MTase activity. Our study thus reveals an additional function for MERS-CoV nsp14 ExoN, which apparently is critical for primary viral RNA synthesis, thus differentiating it from the proofreading activity thought to boost long-term replication fidelity in MHV and SARS-CoV. IO_SCPLOWMPORTANCEC_SCPLOWThe bifunctional nsp14 subunit of the coronavirus replicase contains 3-to-5 exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) domains. For the betacoronaviruses MHV and SARS-CoV, the ExoN domain was reported to promote the fidelity of genome replication, presumably by mediating some form of proofreading. For these viruses, ExoN knockout mutants are alive while displaying an increased mutation frequency. Strikingly, we now established that the equivalent knockout mutants of MERS-CoV ExoN are non-viable and completely deficient in RNA synthesis, thus revealing an additional and more critical function of ExoN in coronavirus replication. Both enzymatic activities of (recombinant) MERS-CoV nsp14 were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.

Genome-wide mapping of therapeutically-relevant SARS-CoV-2 RNA structures (preprint)

SARS-CoV-2 is a betacoronavirus with a linear single-stranded, positive-sense RNA genome of [~]30 kb, whose outbreak caused the still ongoing COVID-19 pandemic. The ability of coronaviruses to rapidly evolve, adapt, and cross species barriers makes the development of effective and durable therapeutic strategies a challenging and urgent need. As for other RNA viruses, genomic RNA structures are expected to play crucial roles in several steps of the coronavirus replication cycle. Despite this, only a handful of functionally conserved structural elements within coronavirus RNA genomes have been identified to date. Here, we performed RNA structure probing by SHAPE-MaP to obtain a single-base resolution secondary structure map of the full SARS-CoV-2 coronavirus genome. The SHAPE-MaP probing data recapitulate the previously described coronavirus RNA elements (5' UTR, ribosomal frameshifting element, and 3' UTR), and reveal new structures. Secondary structure-restrained 3D modeling of highly-structured regions across the SARS-CoV-2 genome allowed for the identification of several putative druggable pockets. Furthermore, [~]8% of the identified structure elements show significant covariation among SARS-CoV-2 and other coronaviruses, hinting at their functionally-conserved role. In addition, we identify a set of persistently single-stranded regions having high sequence conservation, suitable for the development of antisense oligonucleotide therapeutics. Collectively, our work lays the foundation for the development of innovative RNA-targeted therapeutic strategies to fight SARS-related infections.

Suramin inhibits SARS-CoV-2 infection in cell culture by interfering with early steps of the replication cycle (preprint)

The SARS-CoV-2 pandemic that originated from Wuhan, China, in December 2019 has impacted public health, society and economy and the daily lives of billions of people in an unprecedented manner. There are currently no specific registered antiviral drugs to treat or prevent SARS-CoV-2 infections. Therefore, drug repurposing would be the fastest route to provide at least a temporary solution while better, more specific drugs are being developed. Here we demonstrate that the antiparasitic drug suramin inhibits SARS-CoV-2 replication, protecting Vero E6 cells with an EC50 of [~]20 {micro}M, which is well below the maximum attainable level in human serum. Suramin also decreased the viral load by 2-3 logs when Vero E6 cells or cells of a human lung epithelial cell line (Calu-3) were treated. Time of addition and plaque reduction assays showed that suramin acts on early steps of the replication cycle, possibly preventing entry of the virus. In a primary human airway epithelial cell culture model, suramin also inhibited the progression of infection. The results of our preclinical study warrant further investigation and suggest it is worth evaluating whether suramin provides any benefit for COVID-19 patients, which obviously requires well-designed, properly controlled randomized clinical trials.

SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology (preprint)

The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of nonstructural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (Remdesivir, Alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.

Alpha-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication (preprint)

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued structure-based design of peptidomimetic -ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease:inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the -ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against Middle East Respiratory Syndrome coronavirus.